The cell walls of unicellular organisms and of plants mainly consist of polsaccharides, partly associated with proteins. Important functions are: Rigidity, physical protection of the cells, osmotic stability, selective permeability support, cell/cell adhesion, binding of compounds and extracellular enzyme support. Since the polysaccharides form a three dimensional network, cell walls may serve as a resource for particles that maintain certain useful properties of the native cell wall such as selective adsorption capacity. The final properties of the particles depend on the starting material (e.g. yeast strain or other microbial or plant cells) and on the level of preservation of the structural integrity during isolation.
Therefore, a prerequisite for the isolation of functional particles are non-denaturing isolation procedures.
In the following the structure and composition of the yeast cell wall, an important potential resource of said particles, is outlined.
The precise structure and composition of the yeast cell wall strongly depends on the type of yeast strain and culture conditions (R. Bonaly, H. Moulki, A Touimi Benjellouen, M. Pierrefitte, Biochhn. Biophys Acta 244,484 (1971)). A shortage of amino acids, for example, reduces the protein content in the cell wall. Yeasts are unicellular organisms with a rigid cell wall made of polysaccharides. The cell shape is oval to round with an average diameter of 5-13 μm. The cell walls show a thickness of about 70 nm and account for 15-25% of the yeast dry weight (J. S. D. Bacon, V. C. Farmer, D. Jones, I. F. Taylor, Biochem J. 114,557 (1969)). As mentioned, the overall composition of the cell wall varies and depends on the special strain and on culture conditions. This forms the basis for the isolation of a great number of cell wall particles with different properties.
In general, the main components of the yeast cell wall are mannan (typically about 30% by weight), glucan (also about 30% by weight), protein (15% by weight), lipids (about 10% by weight) and chitin (about 2% by dry weight). The latter is exclusively located in the budding scar of the yeasts.
The Mannoprotein Component
By definition mannan, is a polymer that is exclusively composed of mannose units. In yeasts, mannan is associated with protein in both, the external surface of the yeast cell wall, as a muscigenous polysaccharide and in the inner cell membrane. It accounts for 20-50% of the dry weight (C. E. Ballou, Adv. Microbiol. Physiol. 14, 93 (1976). Mannan is linked to a core-peptide chain as an oligomer or polymer (R. Sentandreu, D. H. Northcote, Biochem J. 109,419 (1968)). The complex consists of 5-50% proteins. Oligomeric mannan is bonded directly to serine and threonine (R. Santandreu, D. H. Northcote, Carbohydr. Res. 10, 584 (1969)) whereas polymeric mannan is bonded to aspargine via N-acetylglucosamine. The many individual aspects relating to the mannoprotein complex, including that the mannose units are linked by α-1,6, α-1,2 and α-1,3-linkages were compiled and reviewed by Ballou et al. (C. E. Ballou, Adv. Microbiol. Physiol. 14, 93 (1976); C. E. Ballou, Adv. Enzymol. 40, 239 (1974)).
The Glucan Component
Glucan is a glucose polymer and accounts for 30-60% of the dry weight. The majority of the polyglucoside is linked via β-1,3 glycosidic linkages and only 10-20% by β-1,6 glycosidic linkages (S. Peat, J. R. Turvey, J. M. Evans, J. Chem. Soc. 3868 (1958)). If glucan is treated with approximately 3% caustic soda at 75° C., a maximum of one-third of the glucan is solubilized (J. S. Bacon, V. C. Farmer, D. Jones, Biochem. J. 114, 557(1969)). Consequently the glucan is divided into (1) an alkali insoluble fraction (glucan A), and (2) an alkali soluble fraction (glucan B) (G. H. Fleet, D. J. Manners, J. Gem Microbiol. 94, 180 (1976)).
Glucan A accounts for 80-85% of the cell wall glucan and consists primarily of β-1,3, glycosidic linkages as well as of about 3% β-1,6 glycosidic linkages. 80-85% of the glycosidic linkages of glucan B (15-20% of the total glucan) are β-1,3 and 8-12% are β-1,6 glycosidic linkages 3-4% of the glucose units are branchings. The β-1,6 glycosidic linkages are selectively hydrolysed by acetylosis. It is proposed that the β-1,3 glucan chains are linked via β-1,6 intermediate chains (J. S. D. Bacon, V. O. Farmer, D. Jones, Biochem. J. 114, 557 (1969)). Using electron microscopy it was possible to demonstrate a fibrillar structure for the β-1,3 component and an amorphous structure of the 1,6 component (M. Kopecká, J. Basic Microbial. 25,161 (1985)).
Chitin and Lipid Components
Chittin (N-acylated poly-glucosamine) is located exclusively in the budding scars, where it forms a ring (E. Cabib, B. Browers, J. Biol. Chem. 246, 152 (1971)). As a lipid compound dolichol phosphate was isolated from the cell walls (P. Jung, W. Tanner, Eur. J. Biochem. 37, 1 (1973)). The rest of the lipid component consists of glycerol esters of various fatty acids.
The Structure of the Yeast Cell Wall
Electron microscopic investigation of the process of biosynthesis and assembly of the glucans in Candida albicans reveals the development of the fibrous network of the cell wall. The triple helices which appear as microfibrils with a diameter of approx. 2 nm are self-assembled end-to-end and side by side and are twisted together leading to fibrils of 4-8 nm in diameter. These fibrils finally associate to flat ribbon-shaped bundles, 8-16 nm thick and 100-200 nm wide and thus form the basic network structure of the cell wall. The interfibrillar spaces of the network at this stage have dimensions of about 100-200 nm and most likely mark the origin of the pores which are present in the cell wall at the final stage and which constitute the structural basis for their ability to adsorb compounds with great significance in a large number of different areas. They are gradually filled with the additional components and manno-proteins which are known to form anchors to the membrane lipids.
Isolation of Yeast Cell Wall Components
Fractionation of the cell walls, as e.g. of Saccharomyces cerevisiae starts either from whole cells or from cell walls e.g. obtained by autolysis; both starting materials may be used in dry or wet form. In some cases the cells or cell walls are pre-treated mechanically (by sonification or by treatment with glass beads). The starting material as well as the mechanical disruption greatly influence the purity of the resulting fraction. A large number of different methods were reported for the isolation of cell wall components (F. M. Klis, Yeast 10, 851 (1994)). They can be grouped (1) in methods for the isolation of mannoprotein, and (2) in methods for the isolation of glucan.
A common reagent of chemical methods for the isolation of mannoprotein is sodium hydroxide of varying concentrations and using a wide range of temperatures and treatment times (Int. Patent WO 94/04163 (1994); D. L. Williams, R. B. McNamee, E. L. Jones, H A. Pretus, H. E. Ensley, I. Williams, N. R. DiLuzio, Carbohydr. Res. 219, 203 (1991)). Depending on the reaction conditions, such treatments also solubilize more or less glucan (see above definition of soluble and insoluble glucan), In some cases, organic bases like ethylene diamine and buffers like citrate salts find application to solubilize mannoproteins (R. Sentandreu, D. H. Northcote, Biochem. J. 109, 419 (1968); T. Nakajima, C. Ballou, J. Biol. Chem. 249, 7679 (1974)). Extraction with a 2% boiling sodium-dodecyl-sulfate (SDS) in the presence or absence of reducing agents, like mercaptoethanol, represents a widely used approach to free gluten from mannoproteins and other proteins (E. Valentin, E. Herrero, F. I. J. Pastor, R. Sentandreu, J. General Microbiol. 130, 1419 (1984); F. I. J. Pastor, E. Valentin, E. Herrero, R. Sentandreu, Biophys. Acta 802, 292 (1984)). Treatment of whole cells with pure water at temperatures of up to 135° C. was also applied, yielding a highly contaminated mannoprotein fraction (S. Peat, W. J. Whelan, T. E. Edwards, J. Chem. Soc. 29 (1961); N. Shibata, K. Mizugami, S. Susuki, Microbiol. Immunol. 28, 1283 (1984); Y. Okubo, T. Ichikawa, S Susuki, J. Bact. 136, 63 (1978)).
Enzymatic methods were alternatively used for releasing the manno-proteins. For this purpose, proteases and glucanases are used, acting on the protein part of the mannan or the glucan fixing the mannoprotein (β-1,6 glucan).
The mannan-free glucan is further purified by procedures that include acid treatment such as acetic acid or HCI.
The summarized chemical procedures for isolation and purification of cell wall components will more or less affect the nativity of the polymers, which is primarily reflected in the occurrence of increased amounts of soluble glucan and in a disturbance of the structure of the insoluble glucan fraction. It is especially the latter negative impact of existing glucan isolation procedures that make the insoluble glucan less suitable for adsorbent applications. When such chemical treatments are used under milder conditions, the pores of the glucan skeleton are not properly activated, i.e. freed from physically or chemically bound pore filling material. This also yields insoluble glucan not optimal for adsorption.